Burns
Volume 33, Issue 1 , Pages 72-80, February 2007

Sepsis and burn complicated by sepsis alter cardiac transporter expression

  • Cherry Ballard-Croft

      Affiliations

    • Department of Surgery, University of Kentucky, Lexington, Kentucky, USA
    • ,
  • David L. Maass

      Affiliations

    • Department of Surgery, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, Texas, TX 75390-9160, USA
    • ,
  • Patricia J. Sikes

      Affiliations

    • Department of Surgery, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, Texas, TX 75390-9160, USA
    • ,
  • Jureta W. Horton

      Affiliations

    • Department of Surgery, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, Texas, TX 75390-9160, USA
    • Corresponding Author InformationCorresponding author. Tel.: +1 214 648 3762; fax: +1 214 648 8420.

Accepted 19 June 2006.

Article Outline

Abstract 

Sepsis alone and burn complicated by sepsis produce significant cardiac dysfunction. Since calcium handling by the cardiomyocyte is essential for cardiac function, one mechanism for cardiac abnormalities may be calcium dyshomeostasis. We hypothesized that sepsis and burn plus sepsis alter cardiac calcium transporter expression. Sprague-Dawley rats were divided into: (1) control, (2) sepsis (intratracheal S. Pneumoniae, 4×106 CFU), and (3) burn (40% TBSA) plus sepsis. Myocyte [Ca2+]i and [Na+]i were quantified with Fura-2 AM and SBFI, respectively. Western blot analysis of rat hearts used antibodies against the sarcoplasmic reticular Ca2+ ATPase (SERCA), the L-type calcium channel, the Na+/Ca2+ exchanger or the Na+/K+ ATPase. RESULTS: Sepsis in the presence and absence of burn trauma increased [Ca2+]i and [Na+]i. SERCA expression was decreased in the sepsis and burn plus sepsis groups while calcium channel expression was transiently increased in these sepsis groups. Na+/Ca2+ exchanger expression exhibited a biphasic pattern of altered expression. Sepsis and burn plus sepsis reduced Na+/K+ ATPase protein levels. These data suggest that altered transporter expression produce cardiomyocyte calcium and sodium loading and may contribute to sepsis-mediated cardiac contractile dysfunction.

Keywords: SERCA, L-type calcium channels, Na, K-ATPase, Fluorescent indicators Fura 2AM and SBFI, Intratracheal Streptococcus pneumoniae, Rat model

 

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1. Introduction 

Nearly half of all burn patients in intensive care units develop sepsis. Since this condition is associated with mortality rates as high as 40%, the incidence of burn trauma complicated by sepsis is a serious healthcare issue. Significant cardiac dysfunction occurs in sepsis and burn trauma which is often the cause of morbidity/mortality [1]. Thus, determining the underlying mechanism of cardiac abnormalities in sepsis and burn complicated by sepsis is imperative.

Since the maintenance of cardiomyocyte calcium homeostasis is essential for normal cardiac function, alterations in calcium handling may play a role in the cardiac depression associated with sepsis and burn injury. Although we and others have shown that cardiomyocyte calcium levels are elevated in sepsis and burn trauma [2], [3], [4], [5], the mechanism for this calcium loading has not been fully explored. We further showed that drugs targeting the regulation of intracellular calcium improved cardiac function after both sepsis and burn, decreased cardiac injury, and returned cardiomyocyte calcium to normal levels [2], [4], [5]. In myocardial disease states such as ischemia/reperfusion and heart failure, cardiac contractile deficits are accompanied by changes in cardiac calcium transporter expression [6], [7], [8], [9], [10]. While we previously showed that alterations in calcium transporter expression also occur in burn [11], the expression of these transporters in sepsis and burn complicated by sepsis has not been examined. Therefore, we hypothesized that cardiomyocyte calcium accumulation may occur in sepsis and burn complicated by sepsis through changes in the expression of critical myocardial calcium transporters.

The calcium transporters responsible for calcium influx into the cytosol during cardiac contraction are the sarcoplasmic reticulum ryanodine receptor (SR calcium release channel), the sarcolemmal L-type calcium channel, and the Na+/Ca2+ exchanger [8]. The trigger calcium for activation of calcium release from intracellular stores is derived from the L-type calcium channel while the SR calcium release channel supplies the calcium for myofilament activation.

The calcium transporters that extrude calcium from the cytosol during cardiac relaxation include the sarcoplasmic reticulum Ca2+ ATPase (SERCA), Na+/Ca2+ exchanger and sarcolemmal Ca2+ ATPase. In the rat heart, 92% of the activator calcium is extruded from the cytosol by SERCA with the Na+/Ca2+ exchanger removing 28% and the Ca2+ ATPase 1% of the calcium not extruded by the SR [12], [13]. Thus, reuptake of calcium via the SR Ca2+ ATPase is the main mechanism for calcium removal from the cytosol during relaxation in the rat heart.

In this study, we show that sepsis and burn complicated by sepsis decreased the SR Ca2+ ATPase and the Na+/K+ ATPase protein levels and increased L-type Ca2+ channel expression. Alterations in cardiac transporter protein expression in sepsis and burn plus sepsis preceded the increase in cardiomyocyte calcium, suggesting that changes in cardiac transporter protein levels may play a significant role in cardiomyocyte calcium loading.

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2. Methods 

2.1. Experimental animals 

Adult Sprague Dawley rats (Harlan Laboratories, Houston, TX) weighing 325–360g were used throughout the study. Animals were allowed 5–6 days to acclimate. Commercial rat chow and tap water were available at will throughout the experimental protocol. All work described herein was approved by The University of Texas Southwestern Medical Center Institutional Animal Care and Research Advisory Committee, and was performed according to guidelines outlined in the “Guide for the Care and Use of Laboratory Animals” as published by the American Physiological Society.

2.2. Burn procedure 

Rats were deeply anesthetized with methoxyflurane and secured in a constructed template device as described previously [2]. The skin exposed through the template was immersed in 100°C water for 12s on each side to produce a full thickness dermal burn over 40% total body surface area (TBSA). This burn technique produces complete destruction of the underlying neural tissue. After immersion, the rats were immediately dried and each animal was placed in an individual cage. All burned animals received standard fluid resuscitation consisting of 4ml/kg/%burn lactated Ringer's solution with one-half of this calculated volume given intraperitoneally immediately after completing the burn injury and the remaining volume given 8h postburn. Animals not given a burn served as appropriate controls.

2.3. Induction of sepsis 

Rats were anesthetized with methoxyflurane and placed in a supine position. The area over the trachea was prepared with a surgical scrub (povidone-iodine, betadine), and a midline incision was made over the trachea. The trachea was identified and isolated via blunt dissection. A 0.5ml aliquot of either sterile endotoxin-free PBS or bacterial suspension (S. pneumoniae, 4×106 CFU) was injected directly into the trachea. After closing the wound with surgical staples, the animals were placed on a 30° incline to ensure accumulation of the injected fluid into the lungs [1].

2.4. Experimental groups 

The rats were randomly divided into four major experimental groups: (1.) control (n=8), (2.) sepsis alone (n=7–8), (3.) burn complicated by early sepsis (n=7–8), (4.) burn complicated by late sepsis (n=7–8). Control rats received either a PBS injection alone or in combination with a sham burn. Hearts from the sepsis alone rats were harvested at 2, 4, 8, and 24h post bacterial injection. In the burn complicated by early sepsis group, the rats were subjected to a burn and 48h later, sepsis was induced as described previously. The burn complicated by late sepsis group consisted of rats receiving a burn injury, and sepsis was induced at 72h post burn. Hearts from the two burn complicated by sepsis groups were harvested at 2, 4, 8, and 24h after sepsis induction.

2.5. Cardiomyocyte isolation 

Adult rat cardiomyocytes were isolated as described previously [11]. Briefly, the heart was removed and placed in a Petri dish containing ice cold (4°C) heart medium (113mM NaCl; 4.7mM KCl; 0.6mM KH2PO4; 0.6mM Na2HPO4; 1.2mM MgSO4; 12mM NaHCO3; 10mM KHCO3; 20mM d-glucose; 0.5× MEM (minimum essential medium) amino acids [50X, Gibco/BRL 11130-051]; 10mM Hepes; 30mM taurine; 2.0mM carnitine; and 2.0mM creatine). Hearts were cannulated via the aorta and perfused with the heart medium (composition described above) at a rate of 12ml/min for a total of 5min in a non-recirculating mode. Perfusion was then changed to a re-circulating mode and enzymatic digestion was accomplished with a digestion solution which contained 34.5ml of heart medium described above plus 50mg of collagenase II (Worthington 4177, Lot# MOB3771), 50mg BSA (bovine serum albumin), Fraction V (Gibco/BRL 11018-025), 0.5ml trypsin (2.5%, 10X, Gibco/BRL 15090-046), 15μM CaCl2. This solution was re-circulated through the heart at a flow rate of 12ml/min for 20min. All solutions perfusing the heart were maintained at a constant temperature of 37°C. At the end of the enzymatic digestion, the ventricles were removed and mechanically disassociated in 6ml of enzymatic digestion solution containing a 6ml aliquot of 2× BSA solution (2mg BSA, Fraction V to 100ml of heart media). After mechanical disassociation with fine forceps, the tissue homogenate was filtered through a mesh filter into a conical tube. The cells adhering to the filter were collected by washing with an additional 10ml of 1× BSA solution (100ml of heart medium described above and 1gm of BSA, Fraction V). Cells were then allowed to pellet in the conical tube for 20 minutes. The supernatant was removed and pelleted further in BSA buffer with increasing increments of calcium (100μM, 200μM, 500μM, to a final concentration of 1,000μM). After the final pelleting step, the supernatant was removed, and the pellet was resuspended in MEM (prepared by adding 10.8gm 1× MEM, Sigma M-1018, 11.9mM NaHCO3, 10mM Hepes, and 10ml penicillin/streptomycin, 100 X, Gibco/BRL, 1540–122 with 950ml MilliQ water); total volume was adjusted to 1L. At the time of MEM preparation, the medium was bubbled with 95% O2–5% CO2 for 15min and the pH adjusted to 7.1 with 1M NaOH. The solution was then filter sterilized and stored at 4°C until use. At the final concentration of calcium, the cardiomyocyte cell number was calculated and the myocyte viability was determined.

2.6. Cardiomyocyte calcium and sodium measurements 

Cardiomyocytes were loaded with the calcium indicator Fura2-AM for 45min or the sodium-binding benzofurzan isophthalate (SBFI) for 1h at room temperature in the dark. Myocytes were then suspended in 1.0mM calcium containing minimum essential medium (MEM), washed to remove extracellular dye, and placed on a glass slide on the stage of a Nikon inverted microscope. The microscope was interfaced with Grooney™ optics for epi-illumination, a triocular head, phase optics, and 30× phase contrast objective and mechanical stage. Excitation illumination source (300W compact Xenon arc illuminator) was equipped with a power supply. In addition, this InCyt Im 2™ Fluorescence Imaging System (Intracellular Imaging, Cincinnati, Ohio) included an imaging workstation and Intel Pentium Pro200 MHZ based PC. The computer controlled filter changer allowed alternation between the 340 and 380 excitation wavelengths. Images were captured by monochrome charge-coupled device (CCD) camera equipped with a TV relay lens. InCyt Im2™ Image software allowed measurement of intracellular calcium and sodium concentrations from the ratio of the two fluorescent signals generated from the two excitation wavelengths (340nm/380nm); background was removed by the InCyt IM2™ software. The calibration procedure included measuring fluorescence ratio with buffers containing different concentrations of either calcium or sodium as described previously [11]. At each wavelength, the fluorescence emissions were collected for 1-minute intervals and the time between data collection was 1–2min. Since quiescent myocytes were used in these studies, the calcium levels measured reflect diastolic levels.

2.7. Western blot analysis 

Western blots were performed using rat heart tissue (30μg) harvested at several times after sham, sepsis or burn complicated by sepsis treatments. Briefly, frozen rat hearts were homogenized in ice-cold lysis buffer (0.5g tissue/ml) containing 10mM Hepes, pH 7.4, 2mM EDTA, 0.1% CHAPS, 5mM DTT, 1mM PMSF, and one Mini Complete Protease Cocktail Inhibitor tablet per 10ml of compete buffer (Roche Biochemicals, Mannheim, Germany). The homogenized samples were incubated on ice for 30min and centrifuged at 10,000g for 10min at 4°C. Protein concentration was determined by the Bradford assay (BioRad Protein Assay Reagents, Hercules CA).

Antibodies directed against the following calcium and sodium transporters were used: Ca2+ ATPase, L-type Ca2+ channel (Affinity Bioreagents, Golden, CO), Na+/Ca2+ exchanger (Chemicon, Temecula, CA), and the Na+/K+ ATPase (Upstate Biotechnology, Lake Placid, NY). The Ca2+ ATPase antibody is directed against the muscle-specific SERCA 2 isoform. The L-type Ca2+ channel antibody recognizes the alpha 2 subunit of this channel that is associated with the dihydropyridine-binding alpha 1 subunit. The Na+/K+ ATPase antibody recognizes the alpha 1 subunit.

Following protein determination, the samples were separated on a 10% SDS-polyacrylamide gel for the SR Ca2+ ATPase western blots and a 7.5% gel for the L-type Ca2+ channel immunoblots. The protein was then transferred to PVDF membranes (Millipore, Bedford, MA). The membranes were blocked overnight in 5% nonfat milk and probed for 1h with either Ca2+ ATPase (1:300) or L-type Ca2+ channel (1:250) antibodies. For the Na+/Ca2+ exchanger western blots, the membranes were blocked overnight in 3% BSA/1% milk and probed with the exchanger antibody (1:200) for 1 hour. For the Na+/K+ ATPase westerns, a 10% denaturing gel was used to separate myocardial protein, which was then transferred to nitrocellulose. After blocking overnight in 3% non-fat milk, the membrane was probed with Na+/K+ ATPase antibody (0.075μg/ml). After the primary antibody incubation, the blots were washed (20mM Tris, 135mM NaCl, 0.1% Tween, pH 7.6) three times and incubated for 1h with secondary antibody (1:2000, Promega Madison, WI). Bound antibodies were visualized by enhanced chemiluminescence. To control for variations in signal intensities between blots, the same control sample was used on each blot for normalization. Also, three separate immunoblots were used to determine protein expression.

Quantification of the single band density was determined using Quantity One software (Bio-Rad, version 4.4.0, build 36). Briefly, the radiographic film was scanned using a Scanjet 7400c (Hewlett-Packard, Palo Alto, CA). Densitometry was performed by outlining the selected bands with the volume rectangle tool initially set on the control band of interest. Band density was expressed as arbitrary units (AU) per square millimeter.

2.8. Statistical analysis 

All values are expressed as mean±standard error of mean. Analysis of variance was used to assess overall difference among the groups. Levene's test for equality of variance was used to suggest the multiple comparison procedure to be used. If variance equality was suggested, multiple comparison procedures were performed (Bonferroni); if inequality of variance was suggested, Tamhane multiple comparisons were performed. Probability values less than 0.05 were considered statistically significant (analysis was performed using SPSS for Windows, Version 7.5.1).

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3. Results 

Before investigating the mechanism of calcium accumulation in sepsis, we first confirmed that intracellular calcium was elevated in our adult rat models of sepsis and burn complicated by sepsis. Cardiomyocyte calcium was significantly increased 24h after the induction of sepsis compared to control (Fig. 1). Intracellular calcium was also elevated in the 48 and 72h burn plus 24h sepsis groups. Calcium levels were previously shown to return toward sham levels after 48 and 72h burn alone, and cardiac function is back to normal as well [14].

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  • Fig. 1. 

    Intracellular calcium concentration in cardiomyocytes following 24h sepsis, 48h burn (Day2) complicated by 24h sepsis or 72h burn (Day 3) plus 24h sepsis. Myocytes were isolated from 5–6 rats, loaded with Fura 2AM, and fluorescence measured. Approximately 20 cardiomyocytes from each rat were included for study. All values are mean±S.E.M. Asterisk (*) indicates significant difference from control at p<0.05.

Since cardiomyocyte calcium was increased in sepsis and burn plus sepsis groups, we examined protein expression of various cardiac calcium transporters at various time points in our sepsis and burn complicated by sepsis models. We first focused on the cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA), since removal of calcium from the cytosol via this transporter during diastole is critical for normal cardiac function in the adult rat. Expression of the cardiac SERCA protein was increased and decreased by sepsis. At 8h, SERCA expression was elevated, but 24h after sepsis induction expression of this transporter was reduced (Fig. 2A and B). SERCA expression was also decreased at 2, 4, and 24h sepsis plus 48h burn (Fig. 3A and B). Likewise, SERCA protein levels were also decreased at 4 and 24h sepsis in the 72h burn group (Fig. 3C and D).

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  • Fig. 2. 

    (A). Western blot analysis showing increased and decreased SERCA expression at various times after induction of sepsis. Cont=sham. (B). Densitometric analysis of several western blots (N=3–4) at each time period. All values are mean±S.E.M. Asterisk(*) indicates significant difference from control at p<0.05.

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  • Fig. 3. 

    (A). Representative blot depicting the time course of SERCA expression in 48h burns complicated by sepsis. Cont=sham. (B). Densitometric summary of data (N=8). (C). SERCA protein levels in 72h burns complicated by sepsis as measured by western blot analysis. (D). Densitometric summary of data (N=4). All values are mean±S.E.M. Asterisk(*) indicate significant difference from control at p<0.05.

Since a septic insult changed protein expression of SERCA, the contribution of the two main sarcolemmal calcium transport mechanisms to sepsis-mediated myocardial calcium accumulation was next examined. L-type Ca2+ channel expression was transiently increased 1 and 2h after sepsis induction (Fig. 4A and B) but returned to baseline values by 8h post sepsis. Protein levels of this channel were also elevated at 2 and 4h sepsis but returned to baseline by 24h sepsis in the 48 (Fig. 5A and B) and 72h (Fig. 5C and D) burn groups.

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  • Fig. 4. 

    (A). Effects of sepsis on myocardial L-type Ca2+ channel expression as determined by western blot analysis and (B). densitometric analysis of several westerns (N=4–6) at each time post sepsis. All values are mean±S.E.M. Asterisk(*) indicate significant difference from control at p<0.05.

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  • Fig. 5. 

    (A). L-type Ca2+ channel expression in 48h burns plus sepsis groups and (B). the densitometric analysis (N=8). (C). The protein levels of L-type Ca2+ channel following 72h burns complicated by various times of sepsis. (D). Densitometric summary of data. All values are mean±S.E.M. Asterisk(*) indicates significant difference from control at p<0.05.

Expression of the second sarcolemmal calcium transporter, the Na+/Ca2+ exchanger, was also examined. Na+/Ca2+ exchanger protein levels in response to sepsis were biphasic. Exchanger expression was significantly increased at 4h post sepsis, but significantly decreased 24h after septic insult (Fig. 6A and B). In the 48h burn complicated by sepsis groups, Na+/Ca2+ exchanger expression also exhibited a biphasic pattern with decreased exchanger protein levels at 2 and 24h sepsis/48h burn but increased at 4h sepsis/48h burn (Fig. 7A and B). Na+/Ca2+ exchanger expression also was reduced at 2h sepsis/72h burn group with protein levels returning toward baseline by 24h sepsis/72h burn (Fig. 7C and D).

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  • Fig. 6. 

    (A). Sepsis-related changes in myocardial Na+/Ca2+ exchanger protein as determined by western blot analysis and (B). densitometric summary of several (N=3–5) westerns at each time point. All values are mean±S.E.M. Asterisk(*) indicates significant difference from control at p<0.05.

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  • Fig. 7. 

    (A). Expression of the Na+/Ca2+ exchanger in 48h burns complicated by sepsis as measured by western blot analysis and (B). the densitometric summary of the western data (N=3). (C&D). Exchanger protein levels following 72h burns plus sepsis. All values are mean±S.E.M. Asterisk(*) indicates significant difference from control at p<0.05.

Because the Na+/Ca2+ exchanger closely links calcium and sodium levels, we then determined whether cardiomyocyte sodium levels might also be affected by a septic insult. A significant increase in cardiomyocyte sodium levels was observed 24h after sepsis induction. Intracellular sodium levels were also elevated in the 48 and 72h burns complicated by 24h sepsis groups (Fig. 8).

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  • Fig. 8. 

    Time course of sodium accumulation by cardiomyocytes after sepsis or burn complicated by sepsis. Myocytes were prepared from 5–6 rats at each time point. At least 20 myocytes from each rat were included for each time point. All values are mean±S.E.M. Asterisk(*) indicates significant difference from control at p<0.05.

Since sepsis produces sodium loading (Fig. 8), we also examined the effects of sepsis on myocardial Na+/K+ATPase expression. A decrease in the cardiac expression of this transporter was observed 2–24h post sepsis (Fig. 9A and B). Na+/K+ ATPase protein levels were also reduced in the 48h burn complicated by sepsis groups (Fig. 10A and B). Likewise, expression of this ATPase was also diminished at 4 and 24h sepsis plus 72h burn groups (Fig. 10C and D).

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  • Fig. 9. 

    (A). Effects of sepsis on myocardial Na+/K+ ATPase protein expression as measured by western blot analysis and (B). densitometry. All values are mean±S.E.M. Asterisk(*) indicates significant difference from control at p<0.05.

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  • Fig. 10. 

    Western blot analysis and densitometric summary depicting cardiac Na+/K+ ATPase expression in 48h burns complicated by sepsis (A&B) or in 72h burns plus sepsis (C&D). All values are mean±S.E.M. Asterisk(*) indicates significant difference from control at p<0.05.

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4. Discussion 

The present study shows that sepsis and burn complicated by sepsis cause an elevation in cardiomyocyte calcium levels. Furthermore, sepsis-related alterations in the protein levels of several essential myocardial ion transporters preceded calcium loading of the cardiomyocyte, suggesting one mode of calcium accumulation in sepsis and burn plus sepsis insults. These findings are supported by our previous work in which burn increased cardiomyocyte calcium and changed the expression of key cardiac ion transporters [11]. In addition, several investigators also showed that other cardiac disease states promote calcium overload and change myocardial ion transporter expression [6], [7], [8], [9], [10]. However, this study is the first depicting that these cellular events also occur in sepsis and burn complicated by sepsis models.

In this study, a transient increase in L-type calcium channel expression occurred at 2 and 4h following induction of sepsis. The protein levels of this channel also rose in both the 48 and 72h burns complicated by 2 or 4h sepsis. We previously showed that calcium channel inhibitors diminish calcium and sodium loading in burn cardiomyocytes and provide cardioprotection [2], [15]. Consistent with our data, calcium channel blockers also diminish calcium accumulation in septic models [16] and decrease endotoxin-induced mortality [2]. In contrast, burn in the absence of sepsis decreases L-type calcium channel protein levels, indicating that changes in calcium channel expression may be unique for each insult [17], [18]. Nevertheless, our data suggest that a transient elevation in L-type calcium channel expression may play a role in calcium accumulation in sepsis and burn complicated by sepsis.

Besides the L-type calcium channel, the ryanodine receptor also pumps calcium into the cytosol during contraction. The L-type calcium channel supplies the trigger calcium to activate the ryanodine receptor [19]. Thus, ryanodine receptor activity in sepsis may be increased in response to the increased calcium channel expression observed in the present study. Although neither the activity nor the expression of the ryanodine receptor was included in the current study, our preliminary results indicate that ryanodine receptor expression may also be increased at 4 and 8 hr post sepsis (data not shown). Furthermore, our previous work has shown that an early decrease in calcium channel expression following burn injury was accompanied by a corresponding decrease in ryanodine receptor expression [11].

Changes in the transporters that extrude calcium during relaxation may also play a role in intracellular calcium accumulation. In the rat heart, calcium ATPase (SERCA) is a critical determinant of the extent of relaxation [12], [20]. This transporter also modulates cardiac contraction by replenishing SR calcium stores for contraction [21]. In the present study, we report that rat cardiac SERCA protein levels are altered by sepsis and burn complicated by sepsis. In the sepsis alone groups, a biphasic response was observed. The increase in SERCA expression by 8h post sepsis may be in response to the increased expression of the L-type calcium channel at 2 and 4h post sepsis. However, the expression of SERCA was significantly reduced by 24h sepsis. The protein levels of SERCA were also diminished in the burn complicated by sepsis groups. Cardiac SERCA expression is also dramatically reduced in burn trauma [11], heart failure [22], cardiac hypertrophy [17], and ischemia/reperfusion [6]. In addition to alterations in SERCA protein levels, we have previously reported that burn reduces SERCA activity in guinea pigs [23]. Moreover, Wu et al. [4] found that the activity of this calcium transporter is also depressed in septic rats.

Several investigators have attempted to correct the SERCA expression deficit. Del Monte et al. [24] reported that adenoviral gene transfer of cardiac SERCA2a to failing rat hearts restored cardiac calcium homeostasis and improved cardiac function. A higher SR calcium load and a reduction in diastolic calcium levels also occurred in adult rat cardiomyocytes subjected to adenoviral gene transfer of the fast twitch skeletal muscle SERCA1a [20]. Furthermore, overexpression of SERCA 2a causes maximal rates of contraction and relaxation while a dramatic decrease in contraction and relaxation occurs in mice with only one copy of SERCA2a [18]. Since partial restoration of SR calcium ATPase protein levels dramatically improves calcium handling and function of the heart, maintenance of a certain level of SERCA expression is critical for these key cardiac functions [18], [20], [24]. Combined with our current data, this suggests that diminished SERCA expression may promote calcium accumulation in sepsis and burn complicated by sepsis, which may ultimately lead to cardiac dysfunction.

Besides the SR calcium ATPase, the Na+/Ca2+ exchanger and sarcolemmal calcium ATPase are also active during relaxation. Although the Na+/Ca2+ exchanger and sarcolemmal calcium ATPase extrude much less calcium than SERCA [12], [14], the expression/activity of these transporters becomes more important in cardiac abnormalities where SERCA expression is reduced [10]. In the present study, a biphasic Na+/Ca2+ exchanger expression pattern was observed in the sepsis and burn complicated by sepsis groups. An elevation in Na+/Ca2+ exchanger expression occurred 4h after the induction of sepsis and in the 48h burn complicated by 4h sepsis group. This increase in exchanger expression may help to support cardiac relaxation in the face of increased L-type calcium channel protein levels and reduced SERCA expression. The reduction in Na+/Ca2+ exchanger expression in late sepsis and burn complicated by sepsis is consistent with studies by Wang et al. [25] who showed that Na+/Ca2+ exchanger activity was reduced in late stage sepsis. Our exchanger expression results with the sepsis models in the current study are similar to our previous work with burn trauma in which a biphasic pattern of expression was also observed [11]. Since Na+/Ca2+ exchanger expression appears to be dependent on the post injury time point assayed, this might also explain the apparent discrepancy in exchanger protein levels in failing hearts between various groups [10], [17], [18], [26]. Thus, the initial increase in Na+/Ca2+ exchanger expression may contribute to sodium loading while the reduction in protein levels of this exchanger later on may exacerbate the calcium overload in the presence of reduced SERCA expression.

Since the Na+/K+ ATPase is the main transport mechanism for controlling cardiomyocyte sodium levels, changes in its expression and/or activity may alter both the direction and activity of the Na+/Ca2+ exchanger. The electrogenic Na+/K+ ATPase also helps to sustain the normal resting membrane potential [27]. In the present study, sepsis significantly decreased the protein levels of the α1 subunit of the Na+/K+ ATPase at all post sepsis time points examined. Burn complicated by sepsis also reduced the expression of this transporter. These findings are in agreement with other investigators who found an inhibition of the Na+/K+ ATPase following endotoxin administration [28]. Wu et al. [29] also found that the resting membrane potential was depolarized in septic rats, which was attributed to an impairment of the Na+/K+ ATPase. Studies in failing human myocardium also showed that the protein levels of the Na+/K+ ATPase isoforms are diminished [26]. In contrast, our previous studies have shown that the protein levels of this ATPase were elevated in burn injury in the absence of sepsis [11]. Furthermore, Kato et al. [30], also found that expression of the α1 subunits rose in late stage heart failure in hamsters. The apparent discrepancy in expression patterns of the Na+/K+ ATPase in the different cardiac injuries suggests that changes in the protein levels of this ATPase are dependent on the type of injury. Nevertheless, the observed reduction in the expression of the α1 subunit of the Na+/K+ ATPase is most likely responsible for the rise in cardiomyocyte sodium levels observed in the present study.

The alterations in calcium/sodium transporter expression observed in the present study likely represent changes in protein degradation/synthesis. The decrease in protein levels in the burn complicated by sepsis groups is most likely due to degradation of protein or muscle wasting, a widely documented phenomenon in the skeletal muscle of burn patients [31]. Alterations in protein levels at the early sepsis time points may also reflect changes in mRNA stability. In fact, alterations in SERCA and Na+/Ca2+ exchanger mRNA levels occur within 1–2h in ischemia/reperfusion and pressure overload models [6], [32]. Elevated protein expression at 8 and 24 hr post sepsis and in the burn plus sepsis groups may reflect an increase in gene transcription.

While elevated protein expression may increase ion transporter activity because there would be more protein for ion transport, additional transporters may not be active. Likewise, a decrease in protein expression may reduce transporter activity, but the remaining transporters may exhibit increased activity to compensate for diminished expression. Thus, alterations in protein levels do not always translate into changes in activity. Nevertheless, changes in the level of ion transporter protein expression have been correlated with alteration in transporter activity in ischemia/reperfusion and heart failure models [6], [20], [30]. Furthermore, we have previously shown that sarcoplasmic reticular calcium uptake is diminished in burn trauma, and the current paper shows that the expression of this transporter is also reduced [23]. In addition, other investigators have shown that altered expression of SERCA produces a similar change in SR calcium uptake [20], [24]. Since the magnitude of altered calcium/sodium transporter expression in the current study was similar to other studies in which changes in protein expression led to altered transporter activity [20], [24], the changes in calcium/sodium transporter protein levels observed in the present study may be sufficient to alter activity.

In summary, our findings reveal that alterations in the expression of L-type calcium channel, SERCA, Na+/Ca2+ exchanger, and Na+/K+ ATPase all likely contribute to sodium and calcium accumulation in sepsis and burn complicated by sepsis. Data from the present investigation along with prior studies suggest that alterations in calcium handling after sepsis may play a role in cardiac dysfunction. Therefore, strategies designed to prevent the changes in cardiac transporter expression might provide cardioprotection in sepsis, burn complicated by sepsis, and other myocardial diseases.

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References 

  1. Sheeran PW, Maass SL, White DJ, Turbeville TD, Giroir BP, Horton JW. Aspiration pneumonia-induced sepsis increases cardiac dysfunction after burn trauma. J Surg Res. 1998;76:192–199
  2. Horton JW, White DJ, Maass D, Sanders B, Thompson M, Giroir B. Calcium antagonists improve cardiac mechanical performance after thermal injury. J Surg Res. 1999;87:39–50
  3. Thompson M, Kliewer A, Maass D, Becker L, White DJ, Bryant D, et al. Increased cardiomyocyte intracellular calcium during endotoxin-induced cardiac dysfunction in guinea pigs. Pediatric Res. 2000;47:669–676
  4. Wu L-L, Ji Y, Dony L-W, Liu MS. Calcium uptake by sarcoplasmic reticulum is impaired during the hypodynamic phase of sepsis in rat heart. Shock. 2001;15:49–55
  5. Hulsmann WC, Lamers JMJ, Stam H, Breeman WAP. Calcium overload in endotoxemia. Life Sci. 1981;29:1009–1014
  6. Temsah R, Dyck C, Netticadan T, Chapman D, Eliban V, Dhalla NS. Effect of β-adrenoreceptor blockers on sarcoplasmic reticular function and gene expression in the ischemic-reperfused heart. J Pharm & Exp Therap. 2000;293:15–23
  7. Ueyama T, Ohkusa T, Hisamatsu Y, Nakamura Y, Yamamoto T, Yano M, et al. Alterations in cardiac SR Ca2+ release channels during development of heart failure in cardiomyopathic hamsters. Am J Physiol. 1998;274:H1–H7
  8. Bers DM, Perez-Reyes E. Ca channels in cardiac myocytes: structure and function in Ca influx and intracellular Ca release. Cardiovasc Res. 1999;42:339–360
  9. Rannou F, Dambrin G, Marty I, Carre F, Trouve P, Lompre AM, et al. Expression of the cardiac ryanodine receptor in the compensated phase of hypertrophy in rat heart. Cardiovasc Res. 1996;32:258–265
  10. Studer R, Reinecke H, Bilger J, Eschenhagen T, Bohm M, Hasenfub G, et al. Gene expression of the cardiac Na+-Ca2+ exchanger in end-stage human heart failure. Circ Res. 1994;75:443–453
  11. Ballard-Croft C, Carlson D, Maass DL, Horton JW. Burn trauma alters calcium transporter protein expression in the heart. J Appl Physiol. 2004;97:1470–1476
  12. Bers DM. Calcium fluxes involved in control of cardiac myocyte contraction. Circ Res. 2000;87:275–281
  13. Negretti N, O’Neil SC, Eisner DA. Relative contributions of different intracellular and sarcolemmal systems to relaxation in rat ventricular myocytes. Cardiovasc Res. 1993;27:1826–1830
  14. Maass DL, White DJ, Sanders B, Horton JW. Cardiac myocyte accumulation of calcium in burn injury: cause or effect of myocardial contractile dysfunction. J Burn Care Rehabil. 2006;26:252–2595
  15. White DJ, Maass DL, Sanders B, Horton JW. Cardiomyocyte intracellular calcium and cardiac dysfunction after burn trauma. Crit Care Med. 2002;30:14–22
  16. Hotchkiss RS, Karl IE. Calcium: a regulator of the inflammatory response in endotoxemia and sepsis. New Horiz. 1996;4:58–71
  17. Houser SR, Piacentino V, Weisser . Abnormalities of calcium cycling in the hypertrophied and failing heart. J Mol Cell Cardiol. 2000;32:1595–1607
  18. Periasamy M, Huke S. SERCA pump level is a critical determinant of Ca2+ homeostasis and cardiac contractility. J Mol Cell Cardiol. 2001;33:1053–1063
  19. Adachi-Akahane S, Cleemann L, Morad M. Cross-signaling between L-type Ca2+ channels and ryanodine receptors in rat ventricular myocytes. J Gen Physiol. 1996;108:435–454
  20. Chossat N, Griscelli F, Jourdon P, Logeart D, Ragot T, Heimburger M, et al. Adenoviral SERCA1a gene transfer to adult rat ventricular myocytes induces physiological changes in calcium handling. Cardiovasc Res. 2001;49:288–297
  21. Bers DM. Cardiac excitation-contraction coupling. Nature. 2002;415:198–205
  22. Hassenfuss G, Meyer M, Schillinger W, Preuss M, Pieske B, Just H. Calcium handling proteins in the failing human heart. Basic Res Cardiol. 1997;92:87–93
  23. Horton JW, Kaufman TM, White DJ, Mahony L. Cardiac contractile and calcium transport function after burn injury in adult and aged guinea pigs. J Surg Res. 1993;55:87–96
  24. Del Monte F, Williams E, Lebeche D, Schmidt U, Rosenzweig , Gwathmey JW, et al. Improvement in survival and cardiac metabolism after gene transfer of sarcoplasmic reticulum Ca2+ ATPase in a rat model of heart failure. Circulation. 2001;104:1424–1429
  25. Wang X, Yang J, Dong L, Pang Y, Su J, Tang C, et al. Alteration of Na+-Ca2+ exchange in rat cardiac sarcolemmal membranes during different phases of sepsis. Chinese Med J. 2000;113:18–21
  26. Schwinger RHG, Wang J, Frank K, Muller-Ehmsen J, Brixius K, McDonough AS, et al. Reduced sodium pump α1, α3, and β1-isoform protein levels and Na+,K+-ATPase activity but unchanged Na+-Ca+ exchanger protein levels in human heart failure. Circulation. 1999;99:2105–2112
  27. Gick GG, Hatala MA, Chon D, Ismail-Beigi F. Na,K-ATPase in several tissues of the rat: tissue-specific expression of subunit mRNAs and enzyme activity. J Membrane Biol. 1993;131:229–236
  28. Werdan K, Muller-Werdan U. Elucidating molecular mechanisms of septic cardiomyopathy –the cardiomyocyte model. Molec & Cell Biochem. 1996;163–164:291–303
  29. Wu S-N, Lue S-I, Yang S-L, Hsu H-K, Liu M-S. Electrophysiological properties of isolated adult cardiomyocytes from septic rats. Circ Shock. 1993;41:239–247
  30. Kato K, Lukas A, Chapman DC, Dhalla NS. Changes in the expression of cardiac Na+-K+ ATPase subunits in the UM-X7.1 cardiomyopathic hamster. Life Sci. 2000;67:1175–1183
  31. Barrow RE, Dasu MR, Ferrando AA, Spies M, Thomas SJ, Perez-Polo JR, et al. Gene expression patterns in skeletal muscle of thermally injured children treated with oxandrolone. Ann Surg. 2003;237:422–428
  32. Kent RL, Rozich JD, McCollam PL, McDermott DE, Thacker UF, Menick DR, et al. Rapid expression of the Na+-Ca2+ exchanger in response to cardiac pressure overload. Am J Physiol. 1993;265:H1025–H1029

PII: S0305-4179(06)00190-2

doi:10.1016/j.burns.2006.06.009

Burns
Volume 33, Issue 1 , Pages 72-80, February 2007

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